: Simian Virus 40 T antigen has been used extensively to elucidate the functions needed to transform cells in culture. T antigen is sufficient for the transformation of actively growing cells. Nonetheless, which of T antigen's multiple activities are sufficient to confer specific altered growth properties that collectively define a transformed cell remains to be defined fully. Among the activities of T antigen is the ability to increase transcription from the RNA polymerase I-dependent ribosomal gene promoter. Transactivation of ribosomal genes by T antigen may help supply the increased demand for protein synthesis that accompanies transformation. The objective of this research proposal is to determine the contribution that T antigen-mediated transactivation of the ribosomal gene promoter plays in the transformation process. They showed previously that three activities of T antigen are needed to transactivate a core ribosomal gene promoter in transiently transfected cells: a function in the T common region (amino acids 1-82), the nuclear localization signal and amino acids surrounding site, and a contiguous portion of T antigen spanning amino acids 109-626. The experiments describe in this renewal application are designed to determine whether the same T antigen activities are involved in transactivating ribosomal genes in situ, whether transcription is increased on already active ribosomal gene promoters or alternatively involves the reactivation of silent ribosomal genes, and whether transactivation of ribosomal genes correlates with the acquisition of specific transformed cell properties. To accomplish these objectives, three areas of investigation will be pursued. (1) The involvement of additional activities of T antigen such as those contained within the J domain and the impact of the ribosomal gene enhancer on the transactivation by wild type and mutant T antigens will be assessed. (2) Stable cell lines will be generated containing an inducible wild type T and t, large T or small t antigens alone. The activity at the chromosomal ribosomal promoters will be assessed using the silver staining of the nucleolar organizer regions (NORs) and immunoflourescence detection of T antigen association (3). The relationship between transactivation of ribosomal genes and transformation will be addressed by determining whether mutants that retain the ability to transactivate also retain the ability to stimulate dense focus formation and/or anchorage independent cell division whereas those mutants which lost transactivating capacity also lose transforming activity. This proposal is well within the ability and scope of the principle investigator and the facilities of Elizabethtown College. The DNA cloning cytogenetic analysis, DNA transfection and cellular transformation assays will provide a model system for the training of undergraduate students in addition to furthering our knowledge concerning T antigen mediated transformation.